1. Field of the Invention
The present invention relates to a method for analysis of DNA and the like using capillary array electrophoresis and an apparatus for the analysis.
2. Description of the Related Art
Capillary gel electrophoresis is a rapid and highly sensitive analysis method and is noted as a promising method particularly for DNA analysis including DNA base sequence determination (DNA sequencing). In addition, a capillary array apparatus composed of capillary analyzing tubes put side by side has been developed for increasing the analyzing through-put by analyzing a large number of samples in parallel. In such apparatus, samples are held in a titer plate or a sample bottle, electrodes and the ends of capillaries are put into the titer plate or the sample bottle, and the samples are electrically introduced into the capillaries by application of an electric field between the electrodes and the ends of the capillaries.
On the other hand, there is a growing demand for DNA analysis such as DNA sequencing with the progress of Human Genome Project, and the development of an efficient DNA analysis method is desired. A DNA sequencer using a capillary array gel electrophoresis is also a promising apparatus for the DNA analysis. In addition to such an analyzing apparatus, the improvement of the efficiency of sample preparation and injection into the analyzer has become necessary and various pipetting apparatus are on the market. For preparing samples for DNA sequencing or the like, there is usually used a tube having a capacity of 0.5 ml or a 96-well titer plate. Titer plates are suitable for preparing a large number of samples and are widely used. As to the total volume of reagents used in the reactions carried out in the titer plate, the reaction volume is about 10 xcexcl and cannot be reduced because of the reaction efficiency, and titer plates with a capacity of approximately 100-200 xcexcl are widely used. Substantially the whole amount of the reaction products obtained by the use of a titer plate are used for measurement in a DNA sequencer commercialized with a slab gel, but the amount of samples used in a DNA sequencer using capillaries is one hundredth or less as large as the amount of samples used for measurement using the slab gel. This means that there is a great possibility to reduce the amount, therefore the cost, of reagent(s) used in DNA analysis. However, the conventional sequencing reaction procedure is not suitable for this purpose. Thus, a new tool or method is needed wherein a small amount of reagent is consumed and a small amount of products are efficiently introduced into the capillaries.
An object of the present invention is to provide a DNA sample preparation apparatus which permits reduction of the amount of reagents used, cost reduction and reduction of the number of reactions for labor saving; a method for analyzing DNA or the like by using capillaries array electrophoresis in which large-scale base sequence analysis is carried out by the use of prepared DNA samples; and an apparatus for the analysis.
The present invention provides a sample preparation apparatus suitable for a capillary array type electrophoresis apparatus in which the amount of samples consumed is small. In detail, by the use of a vessel having therein a plurality of separate sample preparation reaction areas, a series of different reactions are carried out in a solution in different places, and the reaction products are held near these places. The samples prepared in the sections (the sample preparation reaction areas) are introduced into different capillaries, respectively. It is convenient to make the pitch of the capillaries at the introducing positions same as the pitch of the sections. For example, the bottom of a groove as vessel is composed of a filter-like membrane permeable to water, buffer, etc. (e.g. filter paper or glass filter), avidin is immobilized on the surface of the filter at uniformly spaced positions, and a liquid containing template DNA tagged biotin is passed through the filter to fix the template DNA to the filter. The groove as vessel is filled with a sequencing primer and a reaction solution, and sequencing reactions are carried out. Thus, a large number of samples are prepared at the same time. The products are hybridized with the DNA fixed to the filter. The end of the capillary is brought close to the products and the products are released from the fixed DNA by heating or the like, introduced into the capillary by applying an electric field and analyzed.
In the DNA fixing portions aligned in the bottom of the groove as vessel, 10 to 20 samples can be fixed per cm when the DNA fixing portions are aligned at a pitch of 0.5 mm to 1 mm. Therefore, 50 to 100 samples can be held in a place of 5 cm width and reacted at a time. When the width of the groove is adjusted to 0.3 mm which is substantially the same as the outside diameter of the capillary, the volume of the reaction solution used is 50 mmxc3x970.3 mmxc3x971 mm=15 xcexcl. Thus, 50 to 100 samples can be reacted by one operation by using the reaction solution in an amount usually required for reacting one sample. When the pitch of the DNA-holding portions is made the same as that of the capillaries of the capillaries array, samples can be introduced into the capillaries at the same time. Therefore, it is very convenient. The constitution of the present invention is explained below in further detail.
One aspect (constitution 1) of the present invention is characterized by a DNA sample preparation apparatus comprising a first component (member) for fixing two or more kinds of DNA samples or primers in its different sections, respectively, and a second component (member) having an open area, wherein reactions including DNA complementary strand syntheses are carried out at the same time and independently in the sections in a space formed by the first component (member) and the open area. More specifically, the first component (member) is composed of a filter membrane, and a biotin-tagged DNA or primer, or an antigen-tagged DNA or primer is fixed to each section of the first component (member), whereby the DNA sample or primer is fixed to the first component (member) by utilizing biotin-avidin bonding or antigen antibody reaction.
Another aspect (constitution 2) of the present invention is characterized by an electrophoresis analysis apparatus comprising a DNA sample preparation apparatus of constitution 1, an electrophoresis analyzer and at least two capillaries having a sample-introducing portion at the end in which the reaction products produced in each section of the DNA sample preparation apparatus of constitution 1 are introduced into a capillary and analyzed by electrophoresis. The analysis apparatus is characterized in that the capillaries are filled with a gel or a sol, that the disposition of the sections is substantially the same as that of the sample-introducing portions at the ends of the capillaries, and that the analysis apparatus is equipped with a means for introducing the reaction products into the sample-introducing portion at the end of the capillary.
Further another aspect (constitution 3) of the present invention is characterized by a DNA sample preparation apparatus comprising a sheet-like component (member) having a plurality of small holes formed at regular distance intervals as reaction vessels and a component (member) having an open area. A DNA sample or a primer is fixed on the inner surface of each small hole and DNA polymerase reaction is carried out; the small holes are formed of a porous material; a biotin-tagged DNA or primer, or an antigen-tagged DNA or primer is fixed in each small hole, whereby the DNA sample or primer is fixed in the small hole by utilizing biotin-avidin bonding or antigen antibody reaction; and reaction reagents are injected into a space formed by the sheet-like component (member) and a component (member) having an open area, to be fed to the small holes.
Still another aspect (constitution 4) of the present invention is characterized by an electrophoresis analysis apparatus comprising a DNA sample preparation apparatus of constitution 3, an electrophoresis analyzer and at least two capillaries having a sample-introducing portion at the end in which the reaction products produced in each small hole of the DNA sample preparation apparatus of constitution 3 are introduced into a capillary and analyzed by electrophoresis. The analysis apparatus is characterized in that the capillaries are filled with a gel or a sol, that the disposition of the small holes is substantially the same as that of the sample-introducing portions at the ends of the capillaries, and that the analysis apparatus is equipped with a means for introducing the reaction products into the sample-introducing portion at the end of the capillary.
Still another aspect (constitution 5) of the present invention is characterized by a DNA sample preparation apparatus comprising capillaries each having a DNA sample or primer fixed on the inner surface and a means for feeding a reaction solution to the capillaries, wherein DNA polymerase reactions are independently carried out inside the capillaries, respectively. This apparatus is characterized in that the number of the capillaries is two or more, that different sample DNA""s are fixed in the different capillaries, that the feeding means feeds the reaction solution to the capillaries at substantially the same time, and that a biotin-tagged DNA or primer, or an antigen-tagged DNA or primer is fixed in each capillary, whereby the DNA sample or primer is fixed in the capillary by utilizing biotin-avidin bonding or antigen antibody reaction.
Still another aspect (constitution 6) of the present invention is characterized by an electrophoresis analysis apparatus comprising a DNA sample preparation apparatus of constitution 5, an electrophoresis analyzer and at least two analyzing capillaries having a sample-introducing portion at the end in which the reaction products produced in each capillary of the DNA sample preparation apparatus of constitution 5 are introduced into an analyzing capillary and analyzed by electrophoresis. The analysis apparatus is characterized in that the analyzing capillaries are filled with a gel or a sol, that the disposition of the capillaries is substantially the same as that of the sample-introducing portions at the ends of the analyzing capillaries, whereby the reaction products are introduced into the analyzing capillary and analyzed by electrophoresis, and that the analysis apparatus is equipped with a means for introducing the reaction products into the sample-introducing portion at the end of the analyzing capillary.
Still another aspect (constitution 7) of the present invention is characterized by a DNA sample preparation apparatus comprising a base plate having a plurality of first grooves for fixing two or more kinds of DNA samples or primers to the inner surfaces of the grooves, respectively, and a second groove filled with a reaction solution and communicating with the first grooves, wherein said reaction solution is introduced into the first grooves to be reacted with the two or more kinds of the DNA samples or primers independently at the same time.
Still another aspect (constitution 8) of the present invention is characterized by an electrophoresis analysis apparatus comprising a DNA sample preparation apparatus of constitution 7, an electrophoresis analyzer and at least two analyzing capillaries having a sample-introducing portion at the end in which the reaction products produced in each first groove of the DNA sample preparation apparatus of constitution 7 are introduced into an analyzing capillary and analyzed by electro-phoresis. The analysis apparatus is characterized in that the analyzing capillaries are packed with a gel or a sol, that a portion into which the analyzing capillary can be inserted is formed at the end of each first groove, and that through this portion, the reaction products are introduced into the sample-introducing portion at the end of the analyzing capillary.
Still another aspect (constitution 9) of the present invention is characterized by a DNA sample preparation apparatus comprising a reaction solution vessel capable of accommodating a reaction solution and a plurality of reaction portions where the reactions of the reaction solution fed from the reaction solution vessel with each of a plurality of samples are independently carried out; and an electrophoresis analysis apparatus comprising a DNA sample preparation apparatus of constitution 9, an electrophoresis analyzer and at least two analyzing capillaries in which the reaction products produced in each reaction portion are introduced into an analyzing capillary and analyzed by electrophoresis. The analysis apparatus is characterized in that the analyzing capillaries are filled with a gel or a sol.
According to the present invention, as many as 100 samples can be subjected to sequencing reactions or fragment analysis reactions by using reagents conventionally used for reacting only two or three samples. Furthermore, as the amount of the reagents handled, a xcexcl order amount, a conventional amount is sufficient, so that not only saving but also easy handling of the reagents are possible.